Abstract: Obesity is an overwhelming problem in Western Society, as approximately 30% of the American adult population is overweight. Obesity is dangerous not only because of the increased metabolic costs associated with overall motility, but also because it is a strong risk factor for the development of diabetes and cardiovascular diseases. Two of the most important cell types involved in obesity are the fat cells, themselves (adipocytes), and, also, liver cells (hepatocytes). Increased fat content of the liver, is a harmful disease (fatty liver disease) that often accompanies obesity, and also, infection with the hepatitis C virus. Macrophages also accumulate fat during coronary artery disease and tuberculosis. Within adipoocytes, hepatocytes, and macrophages, lipid droplets manifest as spherical [unreadable]balls[unreadable]of fat (triglyceride) which can be brightly stained utilizing fluorescent lipid-specific staining reagents. Furthermore, certain proteins (e.g., perilipin), associate closely with the lipid droplets and control metabolism of the triglyceride. The overall purpose of our proposed research is to develop reagents and analytical software to enable performance of microscopy assays for investigating the mechanisms that regulate lipid droplet metabolism. We are developing a software program (Vala Science's Thora) to quantify lipid droplets and the proteins that associate with the lipid droplets, in images obtained from adipocytes, hepatocytes, and macrophages. Secondly, we propose to develop novel monoclonal antibodies to lipid-droplet proteins that will be particularly useful in analyzing the expression and cellular localization of the droplet-associated proteins. The phase I research demonstrated the feasibility of the approach. The research proposed in Phase II will refine the methodology to be used in the assays, improve the utility of our software program, and expand the scope of the project to include quantification of lipid droplets and associated proteins in hepatocytes and macrophages as well as adipocytes. The proposed project will generate products relevant to research in obesity, diabetes, fatty liver, hepatitis C, coronary artery disease and tuberculosis. Narrative: Our proposal is for funds to developed new methods to use to help discover new drugs and chemicals that may be beneficial in treating obesity. Our research will help develop a software program that automatically analyzes images of cells from human fat and human livers that are kept in culture and exposed to potential anti-obesity drugs. We are also proposing to develop new staining reagents (monoclonal antibodies) that will help us with these investigations.